[ccp4bb] resolution limit stuck in Refmac5
Hi all, I'm working with good quality relatively complete x-ray diffraction data collected to a resolution limit 2.6 Å from a crystal of a protein with a small molecule ligand bound. I ran MR from 10-4 Å and then did maximum likelihood rigid body refinement in Refmac5 against data from 50-3.5 Å. Now I would like to run restrained refinement from 50-3 Å. The reason for doing this is that, in order to minimize the divergence between R-cryst and R-free during refinement my advisor who, by the way, is forwarding this e-mail for me (and editing it so please don't bash him too mercilessly) suggested I first build in the ligand and newly positioned polypeptide loops and refine against data at a lower resolution limit before opening it up to all the available data. Apparently this has worked well for him in the past (and it has!). The problem is that I'm to the point where I'd like to extend the resolution down to 3 Å during restrained refinement but even if I set the range from 50-3 Å in the ccp4i window refinement only happens from 50-3.5 Å. If I take a step back and do the rigid body refinement from 50-3 Å and then carry out restrained refinement from 50-3 Å it works fine. Why would the limits imposed by rigid body refinement cause the subsequent restrained refinement to be stuck at the rigid body refinement's resolution limits? Thanks for any thoughts, Jon Fleming Graduate Student Structural Biochemistry Laboratory (Huxford Lab) Department of Chemistry & Biochemistry San Diego State University
Re: [ccp4bb] resolution limit stuck in Refmac5
On Fri, Oct 29, 2010 at 5:46 PM, Tom Huxford wrote: > I'm working with good quality relatively complete x-ray diffraction data > collected to a resolution limit 2.6 Å from a crystal of a protein with a > small molecule ligand bound. I ran MR from 10-4 Å and then did maximum > likelihood rigid body refinement in Refmac5 against data from 50-3.5 Å. Now > I would like to run restrained refinement from 50-3 Å. The reason for doing > this is that, in order to minimize the divergence between R-cryst and R-free > during refinement my advisor who, by the way, is forwarding this e-mail for > me (and editing it so please don't bash him too mercilessly) suggested I > first build in the ligand and newly positioned polypeptide loops and refine > against data at a lower resolution limit before opening it up to all the > available data. Apparently this has worked well for him in the past (and it > has!). The problem is that I'm to the point where I'd like to extend the > resolution down to 3 Å during restrained refinement but even if I set the > range from 50-3 Å in the ccp4i window refinement only happens from 50-3.5 > Å. If I take a step back and do the rigid body refinement from 50-3 Å and > then carry out restrained refinement from 50-3 Å it works fine. Why would > the limits imposed by rigid body refinement cause the subsequent restrained > refinement to be stuck at the rigid body refinement's resolution limits? > Did you refine against the file output by Refmac after rigid-body refinement, instead of using the original reflections? -Nat
Re: [ccp4bb] resolution limit stuck in Refmac5
I guess you are using as input mtz the output mtz from the previous cycle? This will be limited to the requested previous resolution.. it is good practice to always use as input the full data - eg mystuff-scala.mtz output from the scala/ctruncate step.. If the input file has the full resolution then your requested limits should be respected.. Unlike your supervisor I would probably have run the rigid body refinement against limited data then gone straight to using thefull resolution available - restrained refinement of B factors works better the higher the resolution, and provides a very useful way of smudging out errors. Wrong bits often have B factors which go through the roof, and it is then very obvious in the maps But there are many ways to kill a goose, and ditto for refinement strategy.. Eleanor On 10/30/2010 01:46 AM, Tom Huxford wrote: Hi all, I'm working with good quality relatively complete x-ray diffraction data collected to a resolution limit 2.6 Å from a crystal of a protein with a small molecule ligand bound. I ran MR from 10-4 Å and then did maximum likelihood rigid body refinement in Refmac5 against data from 50-3.5 Å. Now I would like to run restrained refinement from 50-3 Å. The reason for doing this is that, in order to minimize the divergence between R-cryst and R-free during refinement my advisor who, by the way, is forwarding this e-mail for me (and editing it so please don't bash him too mercilessly) suggested I first build in the ligand and newly positioned polypeptide loops and refine against data at a lower resolution limit before opening it up to all the available data. Apparently this has worked well for him in the past (and it has!). The problem is that I'm to the point where I'd like to extend the resolution down to 3 Å during restrained refinement but even if I set the range from 50-3 Å in the ccp4i window refinement only happens from 50-3.5 Å. If I take a step back and do the rigid body refinement from 50-3 Å and then carry out restrained refinement from 50-3 Å it works fine. Why would the limits imposed by rigid body refinement cause the subsequent restrained refinement to be stuck at the rigid body refinement's resolution limits? Thanks for any thoughts, Jon Fleming Graduate Student Structural Biochemistry Laboratory (Huxford Lab) Department of Chemistry & Biochemistry San Diego State University
Re: [ccp4bb] resolution limit stuck in Refmac5
Hi Jon On 30 Oct 2010, at 00:46, Tom Huxford wrote: Hi all, I'm working with good quality relatively complete x-ray diffraction data collected to a resolution limit 2.6 Å from a crystal of a protein with a small molecule ligand bound. I ran MR from 10-4 Å and then did maximum likelihood rigid body refinement in Refmac5 against data from 50-3.5 Å. Now I would like to run restrained refinement from 50-3 Å. The reason for doing this is that, in order to minimize the divergence between R-cryst and R-free during refinement my advisor who, by the way, is forwarding this e-mail for me (and editing it so please don't bash him too mercilessly) suggested I first build in the ligand and newly positioned polypeptide loops and refine against data at a lower resolution limit before opening it up to all the available data. I personally would not first build in the ligand and refine against low res data... Assuming the ligand is what you're after, I would: (a) leave the ligand out of the time being (b) refine the model using all data (c) build the ligand in (d) refine the model using all data I'm not clear why doing restrained refinement using limited data first should help.. Apparently this has worked well for him in the past (and it has!). The problem is that I'm to the point where I'd like to extend the resolution down to 3 Å during restrained refinement but even if I set the range from 50-3 Å in the ccp4i window refinement only happens from 50-3.5 Å. If I take a step back and do the rigid body refinement from 50-3 Å and then carry out restrained refinement from 50-3 Å it works fine. Why would the limits imposed by rigid body refinement cause the subsequent restrained refinement to be stuck at the rigid body refinement's resolution limits? Are you using the original reflection file? Best Roberto Thanks for any thoughts, Jon Fleming Graduate Student Structural Biochemistry Laboratory (Huxford Lab) Department of Chemistry & Biochemistry San Diego State University --- Dr. Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail roberto.stei...@kcl.ac.uk
